Characterization of organic arsenic residues in livestock and poultry meat and offal and consumption risks

Referring to [16], accurately weigh 0.300 g of the homogenized sample (to 0.001 g), add 5 mL of nitric acid, and cover the tube to stand overnight. The next day, subject the sample to microwave digestion at 190 °C for 30 min, followed by ultrasonic degassing for 5 min. After digestion, rinse the residues in the tube several times with small amounts of deionized water and transfer all rinses into a 25 mL volumetric flask. Dilute to the mark with deionized water, mix thoroughly, and use the solution for the determination of total arsenic content by inductively coupled plasma mass spectrometry (ICP-MS).

1.00 g of chicken sample was weighed and mixed with 5 mL of extraction solvent (0.15 mol/L HNO₃, 0.15 mol/L HNO₃ – 10 % MeOH, or 0.15 mol/L HNO₃ – 20 % MeOH). Extraction was performed using three methods – hot water (80 °C, 120 min), ultrasound-assisted (80 °C, 60 min), and microwave-assisted (80 °C, 30 min) – which were compared to select the optimal method suitable for our laboratory conditions. After extraction, 2 mL of n-hexane was added, mixed thoroughly by vortexing for 2 min, and centrifuged at 10,000 r/min for 5 min. The upper n-hexane layer was discarded, and the lower aqueous layer was filtered through a 0.22 µm membrane prior to HPLC-ICP-MS analysis. Ten As species – including As(Ⅲ), As(V), AsC, AsB, DMA, MMA, ROX, CBAs, NPAA, and ASA – were determined. Inorganic As content was calculated as the sum of As(Ⅲ) and As(V). Meanwhile, the mobile phase and gradient program shown in Table 1 were applied, and three columns – PRP-X100, AS19, and AS11 – were compared to optimize the separation of As species.

The HPLC-ICP-MS analysis was performed under the following conditions: radiofrequency power of 1,400 W, plasma argon flow rate of 16 L/min, auxiliary gas flow rate of 0.6 L/min, carrier gas flow rate of 0.93 L/min, RPQ 0.40, and the mass-to-charge ratio monitored was m/z = 75 (As). High-purity liquid argon was used. Chromatographic analysis was conducted on a Hamilton PRP-x100 column (4 mm × 250 mm, 5 µm) at a flow rate of 1 mL/min. The mobile phases were as follows: A, ammonia phosphate-ammonia nitrate buffer solution (pH 8.5); B, 1 mmol/L ammonium carbonate; C, methanol. The injection volume was 20 µL, and the gradient elution conditions are shown in Table 1.

The analysis of different arsenic species in pork and its organs revealed that arsenic levels in pig liver were significantly higher than those in pork muscle and kidney, though none of the samples exceeded the national maximum limit of 500 μg/kg. The average total arsenic content in pig liver was 44.70 μg/kg, which is 2.57 times and 1.44 times higher than that in pork muscle (17.38 μg/kg) and pig kidney (31.12 μg/kg), respectively.

In terms of arsenic speciation, inorganic arsenic was the predominant contributor. The inorganic arsenic content in pig liver was 21.52 μg/kg, significantly higher than in pig kidney (13.72 μg/kg) and pork muscle(7.41 μg/kg), accounting for 48.1 %, 44.1 %, and 42.6 % of total arsenic content, respectively. Among the organic arsenic species, dimethylarsinic acid (DMA) was the most abundant, with notably higher concentrations in pig liver (18.89 μg/kg) and kidney (13.39 μg/kg) compared to pork muscle (7.69 μg/kg).

In addition, six arsenic compounds – arsenobetaine (AsB), arsenocholine (AsC), arsenic acid (ASA), nitrophenylarsonic acid (NPAA), carbarsone (CBAs), and roxarsone (ROX) – were not detected in any of the pork muscle, liver, or kidney samples. This absence may be attributed to a lack of exposure sources for these compounds in the pigs’ rearing environment, or to the pigs’ metabolic pathways not favoring the formation of species like AsB and AsC. Furthermore, the detection limits of the analytical method may have restricted the identification of trace-level arsenic species. The results are presented in Table 4.

The total arsenic content in chicken liver was 105.65 μg/kg (all values below the national maximum limit of 500 μg/kg), significantly higher than that in chicken kidney (90.57 μg/kg) and chicken muscle (83.83 μg/kg). This distribution pattern (liver > kidney > muscle) is consistent with the findings for pork and its organs and reflects the central role of the liver in arsenic metabolism and methylation [22]. However, the overall arsenic levels in chicken and its organs were notably higher than those in pork, which may be related to differences in metabolic rate, feed composition, or environmental exposure. Previous studies have demonstrated a significant correlation between arsenic levels in poultry feed and those in chicken tissues (e.g., liver, muscle, and heart), suggesting that feed is a primary source of arsenic accumulation in chickens and may contribute to the observed interspecies differences in arsenic levels [23].

Among the arsenic species detected in chicken and its organs, dimethylarsinic acid (DMA) was the predominant form. The concentrations of DMA in chicken liver, kidney, and muscle were 55.18 μg/kg, 51.21 μg/kg, and 32.08 μg/kg, respectively, accounting for 52.2 %, 56.5 %, and 38.3 % of total arsenic content. The high proportion of DMA may be associated with the use of arsenic-based feed additives in poultry farming, which tend to accumulate in the liver and are metabolized into the less toxic DMA. Chicken liver also had the highest concentration of inorganic arsenic at 41.78 μg/kg, followed by chicken kidney (30.61 μg/kg) and chicken muscle (13.38 μg/kg). This suggests that the liver, as a detoxification organ, may accumulate inorganic arsenic during the methylation process [24].

The results also showed the presence of arsanilic acid (ASA) in chicken muscle, while the other five arsenic species – arsenobetaine (AsB), arsenocholine (AsC), nitrophenylarsonic acid (NPAA), carbarsone (CBAs), and roxarsone (ROX) – were not detected in any of the chicken samples. The absence of NPAA, CBAs, and ROX is likely due to their prohibition by the Ministry of Agriculture in 2018 [8], leading to their minimal use. ASA, previously used as a feed additive for growth promotion and antimicrobial purposes, may still be detected due to historical usage or environmental contamination during poultry farming. Although ASA is less toxic than inorganic arsenic, its metabolites (e.g., inorganic arsenic) may pose potential health risks.

The distribution characteristics of arsenic species in livestock and poultry meat and organs were analyzed using chicken and pig as representative samples. As shown in Figure 4a–c, arsenic speciation exhibited distinct tissue-specific patterns in chicken muscle, liver, and kidney. In chicken muscle, arsenic was predominantly present in organic forms, with DMA and ASA being the major species, accounting for 41.78 % and 40.80 % of the total arsenic content, respectively. Inorganic arsenic species were relatively low, with As(Ⅲ) and As(V) accounting for 13.89 % and 3.53 %, respectively. Notably, ASA was detected only in chicken muscle, while it was not found in the liver or kidney. This may be related to tissue-specific distribution and metabolic excretion characteristics, ASA could accumulate in muscle, whereas in the liver and kidney it is rapidly metabolized into DMA or excreted via the kidneys, resulting in concentrations below the detection limit, This observation is also consistent with existing research findings [10]. DMA remained the dominant species at 56.62 %, followed by As(Ⅲ) (36.73 %) and As(V) (6.15 %). Notably, MMA, an intermediate product of arsenic methylation, was present in chicken liver at a very low level (0.50 %), suggesting a relatively high arsenic methylation capacity in the liver [24]. Chicken kidney had the simplest arsenic profile, with only three species detected: DMA, As(Ⅲ), and As(V). Among them, DMA was the dominant species (62.59 %), followed by As(Ⅲ) (34.43 %), and As(V) was present at the lowest level (3.08 %). This pattern may be closely related to the kidney’s primary function in excretion. These results demonstrate significant differences in arsenic speciation among different tissues, which likely stem from the distinct metabolic and detoxification roles of each organ. The consistent predominance of DMA across all tissues suggests that it may be the principal end product of arsenic metabolism in chickens [25].

Figure 4:

Morphological distribution of arsenic in chicken, pork and their livers and kidneys.

Figure 4d–f shows the arsenic speciation distribution in pork muscle, pig liver, and pig kidney. Compared with chickens, pigs exhibited more uniform arsenic distribution patterns across tissues. Except for a trace amount of MMA detected in pig liver (1.39 %), only DMA, As(Ⅲ), and As(V) were found in all tissues, with DMA and As(Ⅲ) as the dominant forms. Specifically, in pork muscle, DMA and As(Ⅲ) accounted for 50.94 % and 46.17 %, respectively, while As(V) was the least abundant at 2.89 %. In pig liver, the proportions of DMA and As(Ⅲ) were nearly equal (46.10 % and 46.31 %, respectively), with a slightly higher As(V) content (6.20 %) than in muscle. In pig kidney, DMA remained the dominant species (49.39 %), followed by As(Ⅲ) (45.51 %), and As(V) accounted for 5.11 %. This distribution pattern is generally consistent with previous studies. Relevant research has indicated that when pigs ingest arsenic through feed or are exposed to environmental arsenic, the element tends to accumulate more in the liver and kidneys rather than in muscle tissue. In terms of speciation, inorganic arsenic and its methylated products are the predominant forms, with DMA often serving as the principal metabolic end product [26].

According to the China Statistical Yearbook (2024) [27], the average daily consumption of livestock meat in urban areas of Zhejiang Province is 0.107 kg/person, and that of poultry is 0.036 kg/person. In rural areas, the average daily consumption is 0.129 kg/person for livestock meat and 0.037 kg/person for poultry. Based on the inorganic arsenic concentrations determined in the samples (Tables 4 and 5), the average levels of inorganic arsenic in pork muscle, pig liver, and pig kidney were 7.41 μg/kg, 21.52 μg/kg, and 13.71 μg/kg, respectively; while in chicken, chicken liver, and chicken kidney, the average concentrations were 13.38 μg/kg, 41.78 μg/kg, and 30.61 μg/kg, respectively. The average body weights of males and females in Zhejiang Province were reported as 71.6 kg and 58.3 kg, respectively, according to the “Body Weight Management Year” report released by the Zhejiang Center for Disease Control and Prevention on October 18, 2024 [28]. Following the benchmark dose lower confidence limit (BMDL_0.5) for inorganic arsenic set by JECFA, which is 3 μg/kg BW/day, the margin of exposure (MOE) values were calculated using Formulas (1) and (2) to evaluate the health risk from inorganic arsenic intake via consumption of livestock and poultry meat, liver, and kidney.

The results showed that all calculated MOE values were much greater than 1, indicating a low health risk. However, considering that livestock and poultry products are only one of many dietary sources of inorganic arsenic, the actual intake risk may be higher. Specifically, the estimated risk was slightly higher in rural populations compared to urban ones, in females compared to males, for livestock products compared to poultry products, and among different tissues, the order of risk was: liver > kidney > muscle.

Detailed data are presented in Tables 6 and 7. Although current MOE values suggest low risk, attention should still be paid to the cumulative effects of multi-source exposure – particularly for high-risk groups such as pregnant women and young children, and for high-risk food items such as animal offal. It is recommended to strengthen food safety supervision, optimize dietary patterns, and promote scientific food processing and cooking methods to further reduce the exposure risk of inorganic arsenic.